The JNK family MAPKs includes JNK1, JNK2 and JNK3 which have been implicated in a wide range of cellular processes, including cell proliferation, apoptosis and cell migration. Recent studies have demonstrated that abnormal activation of the JNK signaling cascade occurs in many diseases, such as prostate cancer, glioma, as well as squamous cell carcinoma (SCC), the most common and invasive form of nonmelanoma skin cancer in the United States. It is not clear, however, whether this JNK induction plays a dominant role in the pathogenesis of these diseases and whether each JNK protein contributes differently. Genetic studies using mouse models have indicated both redundant and opposite roles in epidermal neoplasia for JNK1 and JNK2, the two predominant JNK proteins expressed in keratinocytes. In addition, it has been demonstrated that JNK expression and activity is increased in psoriatic skin and that inhibition of JNK initiates differentiation whereas augmentation of JNK delays cornification and enhances wound healing in mice. Unfortunately, murine models although very informative do not always recapitulate what occurs in human. Because species-specific differences are possible and pharmacological agents targeting JNK signaling are being actively developed, it is essential to determine the role of each JNK protein in a human model. Here we propose to investigate the functional mechanisms of JNK1 and JNK2 in a human tissue model recently developed by our group. Specifically, we will determine the biological effects of JNK1 and JNK2 on epidermal homeostasis and neoplasia. First, we will generate retroviral-mediated RNA interference (RNAi) and gene expression systems to achieve efficient down-regulation or expression of JNK1 and JNK2 in primary human keratinocytes. Second, we will use the genetically modified human keratinocytes to regenerate human skin in organotypic culture in vitro and skin grafts on immune deficient mice to determine effects on epidermal homeostasis. Third, we will investigate whether JNK1, JNK2 or both are required in epidermal neoplasia by using the human SCC tumor model recently established in our group by expression of oncogenic Ras and MKK7, the JNK upstream activator. In summary, the experiments presented in this proposal are aimed to achieve a better understanding of the role of JNK1 and JNK2 using human epithelial tissues. By the end of it, we hope to have defined the functional mechanisms for both JNK1 and JNK2 in epidermal homeostasis and neoplasia. b:This proposal aims to investigate the functional mechanisms of JNK-MAPKs in epidermal homeostasis. It is relevant to human skin diseases associated with abnormal cell growth and differentiation including cancer.